The steady state intracellular concentration of proteins is regulated by the dynamic balance between rates of synthesis and degradation. The major pathway for degradation within eukaryotes is mediated by the multienzyme ATP, ubiquitin-dependent proteolytic pathway in which proteins are targeted for degradation by the 26S proteasome through their covalent conjugation to the 8.6 kDa polypeptide ubiquitin. Recent evidence indicates the ubiquitin/proteasome degradative pathway is required for a number of fundamental regulatory processes including proteolysis of abnormal proteins, mitotic progression, gene transcription and protein processing, developmentally-programmed cell death, the stress response, organelle biogenesis, and the turnover of various oncoproteins, tumor suppressors, and transcription factors. The long range goal of this proposal is to elucidate the enzymology and function of this pathway for potential therapeutic intervention. The immediate goals of this proposal constitute five specific aims: (1) Site-directed mutagenesis of ubiquitin will be utilized to identify key residues on the polypeptide required for its function as a means of mapping the active sites for key enzymes of the pathway; (2) Mutagenesis of specific residues within E1, the first enzyme of ubiquitin conjugation, will be exploited to identify the active site of this enzyme; (3) Deletion/mutation analysis of the major ubiquitin carrier protein E2/14K will be used to identify regions of the enzyme that interact with E1 and E3, the substrate recognition/conjugation enzyme; (4) Mutagenesis of a recently discovered ubiquitin carrier protein E2EPF, suggested to be required developmentally for the terminal differentiation of keratinocytes, will be utilized to identify the site of autoubiquitination proposed to regulate the self- targeting and intracellular concentration of this enzyme, followed by direct test of the hypothesis in cultured human cell lines by transient transfection of appropriate E2EPF mutants; (5) E2-affinity and two-hybrid screening methods will be used to isolate and clone cognate E3 isozymes requiring E2/14K and E2EPF, followed by their expression and kinetic analysis of the mechanisms for the recombinant conjugating enzymes.